The present invention relates to an integrated-chip-type biosensor and a related method for detection of pathogenic substances. The biosensor and method are particularly, but not exclusively, useful in detecting foodborne pathogens such as Listeria monocytogenes. 
Pathogenic bacteria in foods are the cause of 90% of the cases of reported foodborne illnesses. The Centers for Disease Control and Prevention estimate that there 76 million cases of foodborne illnesses each year in the United States, resulting in hospitalization of 325,000 people, 5,500 deaths, and an annular cost of $7 to $23 billion. E. coli O157:H7 and Listeria monocytogenes are the pathogens of most concern. Ground meat containing E. coli O157:H7 is now considered to be an adulterated food while Listeria monocytogenes has emerged as one of the most important food pathogens with a “zero tolerance” criterion for it in ready-to-eat processed (lunch) meats and dairy foods.
The genus Listeria is comprised of six species, L. monocytogenes, L. ivanovii, L. seeligeri, L. innocua, L. welshimeri, and L. grayi. Of these species, only L. monocytogenes is harmful to humans. Consumption of contaminated food may cause meningitis, encephalitis, liver abscess, headache, fever and gastroenteritis (diarrhea) in immunologically challenged individuals and abortion in pregnant women. L. monocytogenes is ubiquitous in nature and can be found in meat, poultry, seafood, and vegetables. Occurrence of this organism could be as high as 32%. In a food sample, L. monocytogenes is often present in close association with other nonpathogenic Listeria species, thereby complicating the specific detection procedures. A successful detection method ideally detects only L. monocytogenes in the presence of overwhelming populations of nonpathogenic Listeria and other background resident bacteria.
The food processing industry annually carries out more than 144 million microbial tests costing $5 to $10 each. About 24 million of these tests are for detection of food pathogens based on biochemical profile analysis, immunogenic tests (such as enzyme linked immuno-sorbent assays or ELISA), and DNA/RNA probes. These tests are reliable but most require two to seven days to complete because of the steps that are needed to resuscitate cells, increase cell numbers or amplify genetic material needed for detection. This time period is too long for real-time detection of contamination in a food plant and is sufficiently long for contaminated food to be formulated, processed, packaged, shipped, and purchased and eaten by the consumer. Current tests require at least several days to confirm presence of Listeria monocytogenes. The number of annual tests is only expected to increase due to heightened consumer concerns about food safety and the requirement of compulsory testing.
In general, diagnostic tools used for detecting or quantitating biological analytes rely on ligand-specific binding between a ligand and a receptor. Ligand/receptor binding pairs used commonly in diagnostics include antigen-antibody, hormone-receptor, drug-receptor, cell surface antigen-lectin, biotin-avidin, substrate/enzyme, and complementary nucleic acid strands. The analyte to be detected may be either member of the binding pair; alternatively, the analyte may be a ligand analog that competes with the ligand for binding to the complement receptor.
A variety of devices for detecting ligand/receptor interactions are known. The most basic of these are purely chemical/enzymatic assays in which the presence or amount of analyte is detected by measuring or quantitating a detectable reaction product, such as a detectable marker or reporter molecule or ligand. Ligand/receptor interactions can also be detected and quantitated by radiolabel assays.
Quantitative binding assays of this type involve two separate components: a reaction substrate, e.g., a solid-phase test strip and a separate reader or detector device, such as a scintillation counter or spectrophotometer. The substrate is generally unsuited to multiple assays, or to miniaturization, for handling multiple analyte assays from a small amount of body-fluid sample.
In recent years, there has been a merger of microelectronics and biological sciences to develop what are called “biochips.” The term “biochip” has been used in various contexts but can be defined as a “microfabricated device that is used for delivery, processing, and analysis of biological species (molecules, cells, etc.).” Such devices have been used, among other things, for the direct interrogation of the electric properties and behavior of cells (Borkholder et al. “Planar Electrode Array Systems for Neural Recording and Impedance Measurements”, IEEE Journal of Microelectromechanical Systems, vol 8(1), pp. 50-57, 1999); impedance-based detection of protein binding to surfaces, antigen-antibody binding, and DNA hybridization (DeSilva et al., “Impedance Based Sensing of the Specific Binding Reaction Staphylococcus Enterotoxin B and its Antibody on an Ultra-thin Platinum Film,” Biosensors & Bioelectronics, vol. B 44, pp 578-584, 1995); micro-scale capillary electrophoresis (Wooley et al., Ultra High Speed DNA Sequencing Using Capillary Electrophoresis Chips,” Analytical Chemistry, vol. 67(20), pp. 3676-3680, 1995); and optical detection of DNA hybridization using fluorescence signals in the commercially available “DNA-chips” (Fodor et al., “Light-directed Spatially Addressable Parallel Chemical Synthesis,” Science, vol. 251, pp. 767-773).
One of the most interesting uses of biochips is for the detection of small quantities of pathogenic bacteria or toxigenic substances in food, bodily fluids, tissue samples, soil, etc. In applications such as the screening of food products for the presence of pathogenic bacteria, it would be beneficial to detect between 100 and 1000 microorganisms per milliliter of sample, with a sample volume of a couple of milliliters. Not counting the fact that bacteria are substantially larger than single biomolecules (˜2 μm vs. ˜10-100Å), 1000 cells are approximately equivalent to a 10−5 femto-moles of cells, which gives an idea of the difficulty in directly detecting such a small number suspended in a volume of 1 or 2 ml, along with large numbers of food debris, proteins, carbohydrates, oils, and other bacteria. Additionally, in many cases the screening technique must be able to discern between viable and dead cells. Many bacteria will not produce toxins when not viable and consequently will not be pathogenic in that state. DNA detection methods, which search for DNA sequences specific to the pathogen of interest, can be extremely sensitive because they rely on the very specific binding of complementary DNA strands, often coupled with Polymerase Chain Reaction (PCR) for amplification. But the detected DNA fragments cannot reveal whether the pathogen was viable or not. These are the main reasons why current methods of detection almost always involve a growth step, in which the bacteria are cultured to increase their numbers by several orders of magnitude. Once the bacteria are amplified to a large number, visual detection of colonies or Enzyme-Linked Immunosorbent Assays (ELISA) confirm their presence in the original sample. Even though bacteria can multiply very rapidly, this amplification by means of extended growth makes conventional detection methods extremely lengthy, taking anywhere from 2 to 7 days. Thus, one of the main goals of micro-scale detection is a reduced time of analysis, on the order of 2 to 4 hours, to be better than the more conventional methods like plate counts and ELISA.
Numerous reports can be found in the literature on biosensors based on the impedimetric detection of biological binding events, or the amperometric detection of enzymatic reactions. (See DeSilva et al., “Impedance Based Sensing of the Specific Binding Reaction Staphylococcus Enterotoxin B and its Antibody on an Ultra-thin Platinum Film,” Biosensors & Bioelectronics, vol. B 44, pp 578-584, 1995; Mirsky et al., “Capacitive Monitoring of Protein Immobilization and Antigen-antibody Reactions on Monomolecular Alkylthiol films on Gold Electrodes,” Biosensors & Bioelectronics, vol. 112(9-10), pp. 977-989, 1997; Berggren et al., “An Immunilogical Interleukine-6 Capacitive Biosensor Using Perturbation with a Potentiostatic Step,” Biosensors & Bioelectronics, vol. 13, pp. 1061-1068, 1998; Van Gerwen et al., “Nanoscaled Impedimetric Sensors for Multiparameter Testing of Biochemical Samples,” Sensors and Actuators, vol. B 49, pp. 73-80, 1998; Hoshi et al., “Electrochemical Deposition of Avidin on the Surface of a Platinum Electrode for Enzyme Sensor Applications,” Analytical Chimica Acta, vol. 289, pp. 321-327, 1994; Jobst et al., “Mass producible Miniaturized Flow Through a Device with a Biosensor Array,” Sensors and Actuators, vol. B 43, pp. 121-125, 1997; Towe et al., “A Microflow Amperometric Glucose Biosensor,” Biosensors & Bioelectronics, vol. 97(9), pp. 893-899, 1997.) Impedimetric detection works by measuring impedance changes produced by the binding of target molecules to receptors (antibodies, for example) immobilized on the surface of microelectrodes. Amperometric devices measure the current generated by electrochemical reactions at the surfaces of microelectrodes, which are commonly coated with enzymes. Both of these methods can be very sensitive, but preparation of the surfaces of the electrodes (immobilization of antibodies or enzymes) is a complex and sometimes unreliable process, they can be prone to drift, and tend to be very sensitive to noise produced by the multitude of species present in real samples (bodily fluids, food, soil, etc.).
Most, if not all, of the above-mentioned devices are not fully integrated biochips, and sometimes lack integrated electrodes and a sealed fluidic path for the injection and extraction of samples. The most common design of these sensors uses thin metal rods or wires as electrodes, immersed in a flow-through cell. And even those devices based on microfabricated biochips either have a fluidic system separately fabricated over the chip, or the samples are dropped over an open reservoir on the chip, or the whole chip is immersed in a vessel containing the fluids. Having a fully closed system permits the incorporation of sample pre-processing steps, like filtering and chromatography, onto the same chip as the detector.
As mentioned earlier, one of the main goals of bacterial sensors is to determine whether the bacterium of interest is indeed live or dead. A technique that has been widely reported to detect the viability of bacteria on a macro-scale relies on measuring the conductance/impedance changes of a medium in which the microbes are cultured. Such a method is recognized by the Association of Official Analytical Chemists International (AOAC) as a standard technique for the detection of Salmonella in food. This is possible because bacterial metabolism changes the electrolyte concentration in the suspension medium, significantly altering the electrical characteristics of the medium.